Detection, identification and differentiation of eubacterial taxa using a hybridization assay

ABSTRACT

The present invention relates to a method for the specific detection and/or identification of  Enterococcus  species, in particular  Enterococcus faecalis  and/or  Enterococcus faecium , using new nucleic acid sequences derived from the ITS (Internal Transcribed Spacer) region. The present invention relates also to said new nucleic acid sequences derived from the ITS region, between the 16S and 23S ribosomal ribonucleic acid (rRNA) or rRNA genes, to be used for the specific detection and/or identification of  Enterococcus  species, in particular of  Enterococcus faecalis  and/or  Enterococcus faecium , in a biological sample. It relates also to nucleic acid primers to be used for the amplification of said spacer region of  Enterococcus  species in a sample.

This application is a continuation of application Ser. No. 10/535,629,filed May 20, 2005 (abandoned) U.S. national phase of internationalApplication No. PCT/EP2003/013905, filed 8 Dec. 2003, which designatedthe U.S. and claims benefit of EP 02447248.2, filed 6 Dec. 2002, theentire contents of each of which are hereby incorporated by reference inthis application.

FIELD OF THE INVENTION

The present invention relates to a method for the specific detectionand/or identification of Enterococcus species, in particularEnterococcus faecalis and/or Enterococcus faecium, using new nucleicacid sequences derived from the ITS (Internal Transcribed Spacer)region.

The present invention relates also to said new nucleic acid sequencesderived from the ITS region, between the 16S and 23S ribosomalribonucleic acid (rRNA) or rRNA genes, to be used for the specificdetection and/or identification of Enterococcus species, in particularof Enterococcus faecalis and/or Enterococcus faecium, in a biologicalsample.

It relates also to nucleic acid primers to be used for the amplificationof said spacer region of Enterococcus species in a sample.

BACKGROUND OF THE INVENTION

The genus Enterococcus includes currently 27 described species. From thehuman clinical point of view, E. faecalis and/or E. faecium are the mostimportant species: E. faecalis, and E. faecium together make up 95% ofall nosocomial enterococcal infections distributed respectively as 80 to90% and 5 to 10%. Occasionally E. casseliflavus and E. gallinarum areisolated.

Enterococci are increasingly recognized as common causes of infectionthat become difficult to treat because of both inherent and acquiredantibiotic resistance.

Effective control of E. faecalis and/or E. faecium within the hospitaland community requires more aggressive measures that include earlierdiagnosis of colonized patients, in other words, that include a step ofscreening.

Moreover, several of the recently described species do not conform tothe phenotypic characteristics used up to now for their identification.It is therefore necessary and urgent to provide more rapid methods ofdetection and/or identification, using probes and/or primers moresensitive and more specific.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide new nucleic acidsequences derived from a particular region of the ITS of Enterococcusspecies, which can be used, for the detection and/or identification ofEnterococcus species, in particular of E. faecalis and/or E. faecium.

The present invention thus provides an isolated nucleic acid moleculeconsisting of SEQ ID NO 1 or 2, the RNA form of said SEQ ID NO 1 or 2wherein T is replaced by U, the complementary form of said SEQ ID NO 1or 2, or any homologue, and the use of said nucleic acid molecule as atarget for the detection and/or identification of Enterococcus species.

An aspect of the present invention relates to new polynucleotides foruse as probes and/or primers, which have as target a particular regionof the 16S-23S rRNA spacer region of Enterococcus species, and whichallow the detection and/or identification of Enterococcus species, inparticular of Enterococcus faecalis and/or Enterococcus faecium.

The present invention thus provides an isolated nucleic acid moleculethat specifically hybridizes to SEQ ID NO 1 or 2, or to the RNA form ofsaid SEQ ID NO 1 or 2 wherein T is replaced by U, or to thecomplementary form of said SEQ ID NO 1 or 2, or to any homologoussequences thereof, or to a fragment of at least 20 contiguousnucleotides thereof, for the detection and/or identification ofEnterococcus species, in particular of Enterococcus faecalis and/orEnterococcus faecium.

Another aspect of the present invention relates to sets of probes forthe detection and/or identification of Enterococcus species, inparticular of Enterococcus faecalis and/or Enterococcus faecium in asample.

Another aspect of the present invention concerns primers allowingspecific amplification of the 16S-23S rRNA spacer region of Enterococcusspecies, in particular of E. faecalis and/or E. faecium.

Another object of the present invention is a composition containing anyof the new sequences of the invention, or any of the new sets of probesand/or primers of the invention; or a combination thereof.

Another object of the present invention is a kit, in which said probesand/or primers are used, for the detection and/or identificationEnterococcus species, in particular of Enterococcus faecalis and/orEnterococcus faecium.

Another object of the present invention is a rapid and reliablehybridization method for detection and/or identification of Enterococcusspecies, in particular of Enterococcus faecalis and/or Enterococcusfaecium.

Another object of the present invention is a hybridization method basedon real time PCR for detection and/or identification of Enterococcusspecies, in particular of Enterococcus faecalis and/or Enterococcusfaecium.

Table Legends

Table 1: list of SEQ IDs

Table 2: primer pairs

Table 3: set of probes

Table 4: Enterococcus species

DETAILED DESCRIPTION OF THE INVENTION

The following definitions serve to illustrate the terms and expressionsused in the different embodiments of the present invention as set outbelow.

The terms “spacer” and “ITS” (Internal Transcribed Spacer) areabbreviated terms both referring to the region between the 16S and 23SrRNA or between the 16S and 23S rRNA genes.

The term “probe” refers to single stranded oligonucleotides orpolynucleotides which have a sequence which is sufficientlycomplementary to hybridize to the target sequence to be detected.

Preferably the probes of the invention are 70%, 80%, 90%, or more than95% homologous to the exact complement of the target sequence to bedetected. These target sequences are either genomic DNA or precursorRNA, or amplified versions thereof.

The probes of the invention can be formed by cloning of recombinantplasmids containing inserts including the corresponding nucleotidesequences, if need be by cleaving the latter out from the clonedplasmids upon using the adequate nucleases and recovering them, e.g. byfractionation according to molecular weight.

The probes according to the present invention can also be synthesizedchemically, for instance by the conventional phospho-triester method.

The term “complementary” nucleic acids as used herein means that thenucleic acid sequences can form a perfect base-paired double helix witheach other.

The terms “polynucleic acid”, “nucleic acid”, and “polynucleotide”correspond to either double-stranded or single-stranded cDNA or genomicDNA or RNA, containing at least 5, 10, 20, 30, 40 or 50 contiguousnucleotides. A polynucleic acid which is smaller than 100 nucleotides inlength is referred to as an “oligonucleotide”.

They can also refer to modified nucleotides such as inosine ornucleotides containing modified groups which do not essentially altertheir hybridization characteristics.

Single stranded polynucleic acid sequences are always represented in thecurrent invention from the 5′ end to the 3′ end.

They can be used as such, or in their complementary form, or in theirRNA form wherein T is replaced by U.

The term “closest neighbor” means the taxon which is known or expectedto be most closely related in terms of DNA homology and which has to bedifferentiated from the organism of interest.

The expression “taxon-specific hybridization” or “taxon-specific probe”means that the probe only hybridizes to the DNA or RNA from the taxonfor which it was designed and not to DNA or RNA from other taxa.

The term taxon can refer to a complete genus or a sub-group within agenus, a species or even subtype within a species (subspecies, serovars,sequevars, biovars . . . ).

The term “specific amplification” or “specific primers” refers to thefact that said primers only amplify the spacer region from theseorganisms for which they were designed, and not from other organisms.

The term “sensitivity” refers to the number of false negatives: i.e. if1 of the 100 strains to be detected is missed out, the test shows asensitivity of (100-1/100) %=99%.

The term “specificity” refers to the number of false positives: i.e. ifon 100 strains detected, 2 seem to belong to organisms for which thetest is not designed, the specificity of the test is (100-2/100) %=98%.

The oligonucleotides or polynucleotides selected as being “preferential”show a sensitivity and specificity of more than 80%, preferably morethan 90% and most preferably more than 95%.

The term “solid support” can refer to any substrate to which apolynucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate, a membrane (e.g. nylon or nitrocellulose) or amicrosphere (bead). Prior to application to the membrane or fixation itmay be convenient to modify the nucleic acid probe in order tofacilitate fixation or improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH₂ groups, SH groups,carboxylic groups, or coupling with biotin, haptens or proteins.

The term “labeled” refers to the use of labeled nucleic acids. Labelingmay be carried out by the use of labeled nucleotides incorporated duringthe polymerase step of the amplification such as illustrated by Saiki etal. (1988) or Bej et al. (1990) or by the use of labeled primers, or byany other method known to the person skilled in the art. The nature ofthe label may be isotopic (³²P, ³⁵S, etc.) or non-isotopic (biotin,digoxigenin, fluorescent dye, biotin, enzyme, etc.).

The term “signal” refers to a series of electromagnetic waves (forexample fluorescence), or changes in electrical current which carryinformation. The signal can be directly visible, or can be made visibleand/or interpretable by different means or devices.

The “sample” may be any biological material. This biological materialmay be taken either directly from the infected human being, or animal,or after culturing or enrichment, or from food, from the environment,etc.

Biological material may be for example expectoration of any kind,broncheolavages, blood, skin tissue, biopsies, lymphocyte blood culturematerial, colonies, etc. Said samples may be prepared or extractedaccording to any of the techniques known in the art.

The Enterococcus species that are clinically relevant in the meaning ofthe present invention are E. faecalis, E. faecium, E. avium, E. cecorum,E. columbae, E. durans, E. flavescens, E. hirae, E. malodoratus, E.mundtii, E. pseudoavium, E. raffinosus, E. casseliflavus, E. gallinarum(Table 4).

The ITS is already known for some Enterococcus species.

In further studies, the full genome sequencing of different Enterococcusspecies has revealed that these organisms contain at 5 or 6 ribosomalRNA operons in their genome.

In particular, within Enterococcus species, E. faecalis strains show twodifferent type of spacer, and E. faecium strains show four differenttypes.

To solve the problems generated by this very high variability, thepresent invention provides a particular region of the ITS, identifiedand delimited for its great advantage of offering a unique targetsequence for the detection and/or identification of all Enterococcusspecies, and in particular of all Enterococcus species clinicallyrelevant, and more particularly of E. faecalis and/or E. faecium.

Indeed, it has been discovered that the target sequence of the inventionare found in all type of spacer of every Enterococcus species, inparticular of every Enterococcus species that are clinically relevant.

This particular region of the ITS, also referred to as the “targetregion” or “target sequence”, can be defined as a nucleic acid moleculeconsisting of SEQ ID NO 1 or SEQ ID NO 2, or as a nucleic acid moleculethat is homologous to SEQ ID NO 1 or 2, their RNA form wherein T isreplaced by U, or their complementary form.

This term “target sequence” covers all the homologous sequences found inthe ITS of any Enterococcus species, said homologous sequences are alsoreferred to herein after as “homologues”. The degree of homology is thenhigher than 75%, generally higher than 80%, and even higher than 90%.

In the framework of this invention, “homologues” are then homologoussequences to SEQ ID NO 1 or 2 or to any fragment thereof, localized inthe ITS region of any Enterococcus species, SEQ ID NO 1 and 2 beingderived respectively from E. faecalis and E. faecium strains.

New polynucleotides for use as probes and/or primers designed from thetarget sequence of the invention for the detection and/or identificationof Enterococcus species are also an object of the invention.

In other words, an object of the invention relates to newpolynucleotides for use as probes and/or primers, which hybridize withthe target sequence of the invention for the detection and/oridentification of Enterococcus species.

In particular, an object of the invention is an isolated nucleic acidmolecule that specifically hybridizes to SEQ ID NO 1 or 2, or to the RNAform of said SEQ ID NO 1 or 2 wherein T is replaced by U, or to thecomplementary form of said SEQ ID NO 1 or 2, or to a fragment of atleast 20 contiguous nucleotides thereof, or to any of their homologues,for the detection and/or identification of Enterococcus species, inparticular of E. faecalis and/or E. faecium

Preferred polynucleotide probes are between about 5 to about 50 bases inlength, more preferably from about 10 to about 25 nucleotides and aresufficiently homologous to the target sequence.

Polynucleotides of SEQ IDs NO 3 to 84 and any of their homologues may beused as probes.

Preferred probes are polynucleotides of SEQ IDs NO 22 to 26, 28 to 43,45 to 65 and 67 to 84, and homologues, in particular polynucleotides ofSEQ IDs NO 28 to 36, 45 to 58, 67 to 84.

Preferred primers of the invention are single stranded DNApolynucleotides capable of acting as a point of initiation for synthesisof the target sequence of the invention. The length and the sequence ofa primer of the invention must be such that they allow to prime thesynthesis of the extension products.

Preferably a primer of the invention is about 5 to about 50 nucleotideslong, preferably about 15 to about 25. Its specific length and sequenceis to be chosen depending on the conditions used such as temperature andionic strength.

Preferred primers of the invention amplify the target sequence. In otherwords, preferred primers of the invention amplify SEQ ID NO 1 or SEQ IDNO 2 and/or homologues.

Preferred primers of the invention are polynucleotides of SEQ IDs NO 3to 10 and 12 to 20, and homologues.

The fact that amplification primers do not have to match exactly withthe corresponding template sequence to warrant proper amplification isamply documented in the literature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction(PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al.,1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-basedamplification (NASBA; Guatelli et al., 1990; Compton, 1991),transcription-based amplification system (TAS; Kwoh et al., 1989),strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992)or amplification by means of Qβ replicase (Lizardi et al., 1988; Lomeliet al., 1989) or any other suitable method to amplify nucleic acidmolecules known in the art.

The preferred polynucleotides of the invention for use as primers or asprobes are listed in Table 1.

Polynucleotides of the invention may differ in sequence from any of thepolynucleotides specified in Table 1, or from any of their homologues,either by addition to or removal from any of their respectiveextremities of one or several nucleotides, or by changing one or morenucleotides within said sequences, or a combination of both, providedthat the equivalents then obtained still hybridize with the targetsequence as the corresponding unmodified polynucleotides. Saidequivalent polynucleotides share at least 75% homology, preferably morethan 80%, most preferably more than 85% homology with the correspondingunmodified polynucleotides.

When using an equivalent of a polynucleotide, it may be necessary tomodify the hybridization conditions to obtain the same specificity asthe corresponding unmodified polynucleotide.

As a consequence, it will also be necessary to modify accordingly thesequence of other polynucleotides when the polynucleotides are to beused in a set under the same hybridization conditions. Thesemodifications can be done according to principles known in the art, e.g.such as those described in Hames B and Higgins S (Eds): Nucleic acidhybridization. Practical approach. IRL Press, Oxford, UK, 1985.

The polynucleotides primers and/or probes of the invention may alsocomprise nucleotide analogues such as phosphorothioates (Matsukura etal., 1987), alkylphosphorothioates (Miller et al., 1979) or peptidenucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or maycontain intercalating agents (Asseline et al., 1984), etc.

The modified primers or probes require adaptations with respect to theconditions under which they are used in order to obtain the requiredspecificity and sensitivity. However the results of hybridization shouldremain essentially the same as those obtained with the unmodifiedpolynucleotides.

The introduction of these modifications may be advantageous in order toinfluence some characteristics such as hybridization kinetics,reversibility of the hybrid-formation, biological stability of thepolynucleotide molecules, etc.

The probes and primers of the invention are used in methods, alsoobjects of the present invention, for the detection and/oridentification of Enterococcus species, in particular of E. faecalisand/or E. faecium.

Detection and/or identification of the target sequence can be performedby using a electrophoresis method, a hybridization method or asequencing method.

A method of the invention for the detection of one or more Enterococcusspecies in a sample comprises the following steps:

-   -   First, and if necessary, the nucleic acids present in the sample        are made available for amplification and/or hybridization.    -   Secondly, and also if necessary, the nucleic acids, if present,        are amplified with one or another target amplification system,        as specified below. Usually, amplification is needed to enhance        the subsequent hybridization signal. However for some samples,        or for some highly sensitive signal-amplification systems,        amplification might not be necessary.    -   Thirdly, the nucleic acids present in the sample or the        resulting amplified product are contacted with probes, and        hybridization is allowed to proceed.    -   Finally, the hybrids are detected using a convenient and        compatible detection system. From the hybridization signals or        patterns observed the presence or absence of one or several        Enterococcus species can be deduced.

The amplification system used may be more or less universal, dependingon the specific application needed.

By using universal primers located in the conserved flanking regions(16S and 23S gene) of the rRNA spacer, the spacer region of most if notall organisms of eubacterial origin will be amplified.

For some applications it may be appropriate to amplify not all organismspresent in the sample but one or several Enterococcus species. This maybe achieved using specific primers located in the target region ofEnterococcus species.

In particular, a method of the invention for detection and/oridentification of Enterococcus species, notably of Enterococcus faecalisand/or Enterococcus faecium, in a sample comprises the steps of:

(i) if need be releasing, isolating and/or concentrating the polynucleicacids in the sample;

(ii) if need be amplifying the 16S-23S rRNA spacer region, or a fragmentcomprising the target sequence, or the target sequence or a fragmentthereof, with at least one suitable primer pair;

(iii) hybridizing the polynucleic acids of step (i) or (ii) with atleast one polynucleotide probe that hybridizes to the target sequence,wherein the target sequence consists of SEQ ID NO 1 or 2 or homologuesthereof, or to their RNA form wherein T is replaced by U, or to theircomplementary form, or a to a fragment of at least 20 contiguousnucleotides thereof,

(iv) detecting the hybrids formed, and

(v) interpreting the signal(s) obtained and inferring the presence ofEnterococcus species and/or identifying the Enterococcus species in thesample.

Preferably, the probes of the inventions hybridize under conditions ofhigh stringency.

Under high stringency conditions only complementary nucleic acid hybridsare formed. Accordingly, the stringency of the assay conditionsdetermines the amount of complementarity needed between two nucleic acidstrands forming a hybrid. Stringency is chosen to maximize thedifference in stability between the hybrid formed with the target andthe non-target nucleic acid.

The hybridization conditions are chosen in such a way that the signal ofhybridization obtained when a polynucleotide of the invention hybridizesspecifically to a target sequence, is different from the signal obtainedwhen said polynucleotide hybridizes to a target sequence in anon-specific manner.

In practice, the different signals may be visualized for example whenits intensity is two, five, ten or more times stronger with a specifichybridization to the target, as compared to non-specific hybridizationto the target sequence, LiPA system for example.

The different signals may also be visualized when different peaks aredrawn in a melting curve analysis, for instance when using a real timePCR method.

The fragment mentioned in the amplification or the hybridization step ofany method of the invention may comprise 20 to 50, 20 to 80 or 20 to 100contiguous nucleotides of SEQ ID NO 1 or 2 or of any homologues.

In one embodiment, a very convenient and advantageous technique for thedetection of target sequences that are possibly present in the sample isthe real time PCR method.

There are different formats for the detection of amplified DNA, notablyTaqMan™ probes, Molecular Beacons probes, or FRET hybridization probes.

Concerning the TaqMan™ probes, a single-stranded hybridization probe islabeled with two components. When the first component, the so-calledfluorescer, is excited with light of a suitable wavelength, the absorbedenergy is transferred to the second component, the so-called quencher,according to the principle of fluorescence resonance energy transfer.During the annealing step of the PCR reaction, the hybridization probebinds to the target DNA and is degraded by the 5′-3′ exonucleaseactivity of the polymerase, for example Taq Polymerase, during theelongation phase. As a result the excited fluorescent component and thequencher are spatially separated from one another and thus afluorescence emission of the first component can be measured (EP B 0 543942 and U.S. Pat. No. 5,210,015).

Concerning Molecular Beacons probes, the probes are also labeled with afirst component and with a quencher, the labels preferably being locatedat different ends of an at least partially self-complementary probe. Asa result of the secondary structure of the probe, both components are inspatial vicinity in solution. After hybridization to the target nucleicacids both components are separated from one another such that afterexcitation with light of a suitable wavelength the fluorescence emissionof the first component can be measured (U.S. Pat. No. 5,118,801).

The Fluorescence Resonance Energy Transfer (FRET) hybridization probetest format is especially useful for all kinds of homogenoushybridization assays (Matthews, J. A. and Kricka, L. J., Anal Biochem169 (1988) 1-25). It is characterized by two single-strandedhybridization probes which are used simultaneously and are complementaryto adjacent sites of the same strand of an (amplified) target nucleicacid. Both probes are labeled with different fluorescent components.When excited with light of a suitable wavelength, a first componenttransfers the absorbed energy to the second component according to theprinciple of fluorescence resonance energy transfer such that afluorescence emission of the second component can be measured only whenboth hybridization probes bind to adjacent positions of the targetmolecule to be detected.

When annealed to the target sequence, the hybridization probes must belocated very close to each other, in a head to tail arrangement.Usually, the gap between the labeled 3′ end of the first probe and thelabeled 5′ end or the second probe is as small as possible, and notablyconsists of about 0 to 25 bases, and preferably of about 1 to about 5bases. This allows for a close vicinity of the FRET donor compound andthe FRET acceptor compound, which is typically 10-100 Angstrom.

Alternatively to monitoring the increase in fluorescence of the FRETacceptor component, it is also possible to monitor fluorescence decreaseof the FRET donor component as a quantitative measurement ofhybridization event.

Among all detection formats known in the art of real time PCR, theFRET-hybridization probe format has been proven to be highly sensitive,exact and reliable (WO 97/46707; WO 97/46712; WO 97/46714). Yet, thedesign of appropriate FRET hybridization probe sequences may sometimesbe limited by the special characteristics of the target nucleic acidsequence to be detected.

As an alternative to the usage of two FRET hybridization probes, it isalso possible to use a fluorescent-labeled primer and only one labeledpolynucleotide probe (Bernard, P. S., et al., Anal. Biochem. 255 (1998)101-7). In this regard, it may be chosen arbitrarily, whether the primeris labeled with the FRET donor or the FRET acceptor compound.

FRET hybridization probes (also called Hybprobes or FRET-probes) canalso be used for melting curve analysis (WO 97/46707; WO 97/46712; WO97/46714). In such an assay, the target nucleic acid is amplified firstin a typical PCR reaction with suitable amplification primers. Thehybridization probes may already be present during the amplificationreaction or be added subsequently. After completion of the PCR-reaction,the temperature of the sample is consecutively increased. Fluorescenceis detected as long as the hybridization probe is bound to the targetDNA. At the melting temperature, the hybridization probe is releasedfrom their target, and the fluorescent signal is decreasing immediatelydown to the background level. This decrease is monitored with anappropriate fluorescence versus temperature-time plot such that thenegative of a first derivative function can be calculated. Thetemperature value corresponding to the obtained maximum of such afunction is then taken as the determined melting temperature of saidpair of FRET hybridization probes.

Point mutations or polymorphisms within the target nucleic acid resultin a less then 100% complementarity between the target nucleic acid andthe FRET probes, thus resulting in a decreased melting temperature. Thisenables for a common detection of a pool of sequence variants by meansof FRET-Hybprobe hybridization, whereas subsequently, different membersof said pool may become discriminated by means of performing meltingcurve analysis.

Instead of FRET hybridization probes, Molecular Beacons mayalternatively be used for melting curve analysis.

Upon the availability of Real-Time PCR and homogenous Real-Time PCRmelting curve analysis, discrimination of certain types of species orstrains became possible using either double stranded DNA binding dyessuch as SybrGreenTMI, or, alternatively, specifically designedhybridization probes hybridizing to different but similar targetsequences.

In the first case, melting temperature of the generated double strandedPCR product has to be determined. Yet, this method has only limitedapplications since few differences cannot be monitored efficiently,because minor sequence variations only result in subtle meltingtemperature differences.

Alternatively, hybridization probes may be used in such a way that themelting temperature of the probe/target nucleic acid hybrid is beingdetermined.

There are different real time PCR platforms such as the ABI/Prism™equipments, and in particular the LightCycler™ apparatus, all based onthe same principle consisting of measuring the light emission,continually monitoring the emission peak during the melt cycle,determining and visualizing the temperatures (melting peaks) at whichthe labeled probes detach from the amplification products. The meltingpeak data are characteristic of a particular [probe:target] sequencebecause mismatches between probe and target affect the kinetics ofmelting, producing different melting peaks for each species of interest.

The LightCycler™ platform offers many advantages and in particular again of time and the possible use of several different sequence-specificfluorescent probe detection systems such as hybridization probes(HybProbes), TaqMan™ probes, Molecular Beacons and biprobes (SYBR GreenI).

In a preferred method of the present invention, the HybProbe system isused, consisting of two adjacent polynucleotide probes derived from thetarget region of the invention, in a head-to-tail orientation, spaced bya few nucleotides, generally 0 to 25, preferably about 1 to about 5. Oneof the probes is labeled at its 3′ end by a donor dye, the other islabeled with an acceptor molecule at its 5′ end, and is phosphateblocked at the 3′ end (to prevent its acting as a primer). The donor dyeis generally fluorescein, and the acceptor molecule generally LC Red640or 705.

The detection of the target sequence of the invention may be achievedalso by an internal labeled PCR strand and a detection probe located onthe opposite strand. The signal is dependent on the spatialapproximation of the dyes, and is dependent on the amount of the target.

When both probes are hybridized to their target sequence the emittedlight of the donor is transmitted to the acceptor fluorophore byFluorescence Resonance Energy Transfer (FRET), and the emittedfluorescence (640 or 705 nm) can be detected. The intensity of theemitted fluorescence increases in parallel with the target DNA, productof the amplification.

The LightCycler probes offer the advantage over the TaqMan™ probes ofnot requiring hydrolysis and, therefore, no additional extension of thePCR times (annealing-elongation ≦12 s). It is therefore possible to takeadvantage of the high-speed thermal cycling of the LightCycler, andcomplete the PCR program in only 45 minutes.

And the most recent generations of this real-time PCR platform are ableto monitor several probes in a single reaction, allowing the detectionand/or identification of different Enterococci, at the species level andalso at lower taxonomical levels.

Moreover, it has been shown that the methods designed for TaqMantechnology can be easily converted to HybProbe technology withequivalent results (Haematologica vol. 85 (12) pp. 1248-1254, December2000).

Therefore another object of the invention relates to sets of at least 2polynucleotide probes, also referred to as HybProbes, both HybProbeshybridizing to the same target sequence, adjacent to each other, with nomore than 25 nucleotides between said 2 HybProbes, preferably with nomore than 10 nucleotides, in particular with no more than 5 nucleotides.

When there are two HybProbes, one is labeled with an acceptorfluorophore and the other with a donor fluorophore of a fluorescenceenergy transfer pair such that upon hybridization of the two HybProbeswith the target sequence, the donor and acceptor fluorophores are within0 to 25 nucleotides of one another, and preferably within 0 to 5nucleotides of one another.

When there are more than two HybProbes, some are labeled with anacceptor fluorophore and the others with a donor fluorophore of afluorescence energy transfer pair such that upon hybridization of theHybProbes with the target sequence, the donor and acceptor fluorophoresare within 0 to 25 nucleotides of one another, and preferably within 0to 5 nucleotides of one another.

For detecting and/or identifying Enterococcus species, in particularEnterococcus species clinically relevant, a set of at least twopolynucleotide probes may be used, said probes hybridizing to SEQ ID NO1 or SEQ ID NO 2, or to the RNA form of said SEQ ID NO 1 or 2 wherein Tis replaced by U, or to the complementary form of said SEQ ID NO 1 or 2,or to homologues, wherein there are no more than 25 nucleotides,preferably no more than 5 nucleotides, between said probes.

A set of probes of the invention may also consist of 3, 4, 5, 6, 7, 8,9, 10, or more, probes, but it preferably consists of 2 to 5 probes, andmore preferably of 3 probes.

The sets of probes listed in Table 3 and their homologues are preferredsets of the invention.

Sets of 2 polynucleotides, one for use as primer, the other for use asprobe, may also be used, both said primer and probe hybridizing to thetarget sequence consisting of SEQ ID NO 1 or 2, of the RNA form of saidSEQ ID NO 1 or 2 wherein T is replaced by U, of the complementary formof said SEQ ID NO 1 or 2, or of any homologues, wherein there are nomore than 25 nucleotides, preferably no more than 5 nucleotides, betweensaid primer and said probe.

The sets of at least 2 polynucleotides of the invention are used inmethods for the detection and/or identification of Enterococcus species,in particular of E. faecalis and/or E. faecium.

A method of the present invention for detection and/or identification ofEnterococcus species in a sample, comprises the steps of:

(i) if need be releasing, isolating and/or concentrating the polynucleicacids in the sample;

(ii) amplifying the 16S-23S rRNA spacer region, or the target sequence,or a part of the spacer comprising the target sequence, or a part of thetarget sequence, with at least one suitable primer pair;

(iii) hybridizing the polynucleic acids with at least one set of atleast two HybProbes that hybridize to the target sequence, wherein thetarget sequence consist of SEQ ID NO 1 or 2, or of the RNA form of saidSEQ ID NO wherein T is replaced by U, or of the complementary form ofsaid SEQ ID NO, or of any homologues, or of a fragment of at least 20contiguous nucleotides thereof;

(iv) detecting the hybrids formed in step (iii);

(v) inferring the presence of Enterococcus species, or identifying theEnterococcus species in the sample from the differential hybridizationsignals obtained in step (iv).

For example, a primer pair used in the amplification step is anycombination of a forward primer consisting of SEQ ID NO 3 to 11 or theirhomologues, and a reverse primer consisting of SEQ ID 12 to 21 or theirhomologues.

For example, a set of 2 or 3 HybProbes used in the hybridization step isany combination of 2 or 3 HybProbes chosen among polynucleotides of SEQIDs NO 22 to 26, 28 to 43, 45 to 65 or 67 to 84 or their homologues,preferably among polynucleotides of SEQ IDs NO 28 to 36, 45 to 56 or 67to 84 or their homologues, provided that the gap between two of saidHybProbes when hybridized to the target sequence is less than 25nucleotides, preferably less than 5 nucleotides.

Preferred sets are mentioned in Table 3.

One of the advantages of the HybProbes system resides in the fact thatit allows the detection of sequence variation, including mutations,polymorphisms and other variant nucleic acid species, based on thefollowing molecular concept: one of the HybProbe is a tightly binding“anchor probe” whereas the adjacent “sensor probe” spans the region ofsequence variation. During melting of the final PCR product, thesequence alteration is detected as a change in the melting temperature(Tm) of the sensor probe.

For example, if the sample contains only SEQ ID NO 1, using Hybprobesthat specifically hybridize to said SEQ ID NO 1 would generate a singlemelting peak. If there is also a homologue in the sample, using the sametwo HybProbes would generate two peaks, as far as there is onemismatched base which generally induces a temperature shift easilyobservable.

Depending on the polynucleotides selected, their Tm and thehybridization conditions, the fluorescence may be measured during theamplification step, generating then amplification curves, or after theamplification step, for a melting curve analysis generating meltingcurves.

Thus the signal obtained may be visualized in the form of amplificationcurves or in the form of melting curves, from which it is possible toinfer the presence of Enterococcus species, and/or to infer which one(s)of the Enterococci are present.

In particular, a method for detection and/or identification ofEnterococcus species in a sample comprises also the steps of

(i) if need be releasing, isolating and/or concentrating the polynucleicacids in the sample, and

(ii) amplifying the target sequence, or a part of it, with a primer pairthat is labeled,

(iii) hybridizing the polynucleic acids with at least one HybProbe thathybridize, adjacent to said labeled primer with less than 25 nucleotidesin between, to SEQ ID NO 1 or 2, or to the RNA form of said SEQ ID NO 1or 2 wherein T is replaced by U, or to the complementary form of saidSEQ ID NO 1 or 2, or to any homologues, or to a fragment of at least 20contiguous nucleotides thereof,

(iv) detecting the hybrids formed, and

(v) inferring the presence of Enterococcus species, and/or identifyingthe Enterococcus species in the sample from the signals obtained in step(iv).

A method of the invention using the HybProbes system, may be adapted forthe detection and identification of Enterococcus faecalis and/orEnterococcus faecium, allowing the distinction of E. faecalis and/or E.faecium from other species.

Then, in the amplification step, suitable primers are primer pairs thatspecifically amplify the target sequence which consists of SEQ ID NO 1or 2, or of the RNA form of said SEQ ID NO 1 or 2 wherein T is replacedby U, or of the complementary form of said SEQ ID NO 1 or 2.

In the hybridization step, the HybProbes should hybridize specificallyto SEQ ID NO 1 or 2, or to the RNA form wherein T is replaced by U, orto the complementary form.

Therefore, E. faecalis and/or E. faecium strains can be unequivocallydistinguished from all other organisms examined by melting curveanalysis.

No relevant signals are obtained with non-Enterococci or human genomicDNA.

Preferred primer pairs used in this particular example are anycombinations of forward primers chosen among SEQ ID NO 3 to 11 or theirhomologues and reverse primers chosen among SEQ ID NO 12 to 21 or theirhomologues.

The sets of HybProbes listed in Table 3 or their homologues are thepreferred sets of HybProbes of the invention. A more preferred set of 3Hybprobes consists of SEQ ID NO 36 or homologues and SEQ ID NO 56 orhomologues and SEQ ID NO 73 or homologues.

The set of HybProbes consisting of SEQ ID NO 36, 56 and 73 is able todetect E. faecalis and/or E. faecium with a high sensitivity.

Each polynucleotide listed in Table 1, corresponding to SEQ ID NO 1 toSEQ ID NO 84 and any of their homologues, may be used in any methods ofthe present invention as a primer and/or as a probe, alone or incombination.

A second embodiment based also on a hybridization method is the LineProbe Assay technique. The Line Probe Assay (LiPA) is a reversehybridization format (Saiki et al., 1989) using membrane strips ontowhich several polynucleotide probes (including negative or positivecontrol polynucleotides) can be conveniently applied as parallel lines.

The LiPA technique, as described by Stuyver et al. (1993) and ininternational application WO 94/12670, provides a rapid anduser-friendly hybridization test. Results can be read within 4 h. afterthe start of the amplification. After amplification during which usuallya non-isotopic label is incorporated in the amplified product, andalkaline denaturation, the amplified product is contacted with theprobes on the membrane and the hybridization is carried out for about 1to 1.5 h. Consequently, the hybrids formed are detected by an enzymaticprocedure resulting in a visual purple-brown precipitate. The LiPAformat is completely compatible with commercially available scanningdevices, thus rendering automatic interpretation of the resultspossible. All those advantages make the LiPA format liable for use in aroutine setting.

The LiPA format is an advantageous tool for detection and/oridentification of pathogens at the species level but also at higher orlower taxonomical levels. For instance, probe-configurations on LiPAstrips can be selected in such a manner that they can detect thecomplete genus of Enterococcus or can identify species within the genus(e.g. Enterococcus faecalis and/or Enterococcus faecium, etc) or can insome cases even detect subtypes (subspecies, serovars, sequevars,biovars, etc. whatever is clinically relevant) within a species.

The ability to simultaneously generate hybridization results with alarge number of probes is another benefit of the LiPA technology. Inmany cases the amount of information which can be obtained by aparticular combination of probes greatly outnumbers the data obtained byusing single probe assays. Therefore the selection of probes on themembrane strip is of utmost importance since an optimized set of probeswill generate the maximum of information possible.

These probes can be applied to membrane strips at different locationsand the result is interpreted as positive if at least one of theseprobes is positive. Alternatively these probes can be applied as amixture at the same location, hereby reducing the number of lines on astrip. This reduction may be convenient in order to make the strip moreconcise or to be able to extend the total number of probes on one strip.

Another alternative approach, in view of its practical benefits, is thesynthesis of polynucleotides harboring the sequences of two or moredifferent probes, referred to as degenerate probes, which then can befurther processed and applied to the strip as one polynucleotidemolecule. This approach would considerably simplify the manufacturingprocedures of the LiPA-strips. For example, probes with nucleotidesequences A and B are both required to detect all strains of taxon X. Inthe latter alternative a probe can be synthesized having the nucleotidesequence AB. This probe will have the combined characteristics of probesA and B.

By virtue of the above-mentioned properties the LiPA system can beconsidered as an efficient format for a hybridization method whereinseveral organisms need to be detected simultaneously in a sample.

However, it should be clear that any other hybridization assay, wherebydifferent probes are used under the same hybridization and washconditions can be used for the above-mentioned detection and/orselection methods. For example, it may be possible to immobilize thetarget nucleic acid to a solid support, and use mixtures of differentprobes, all differently labeled, resulting in a different detectionsignal for each of the probes hybridized to the target. And nowadaysmany different supports are available.

As an example, the procedure to be followed for the detection of one ormore Enterococcus species in a sample using the LiPA format is outlinedbelow:

-   -   First, and if necessary, the nucleic acids present in the sample        are made available for amplification and/or hybridization.    -   Secondly, the nucleic acids, if present, are amplified with one        or another target amplification system, as specified below.        Usually, amplification is needed to enhance the subsequent        hybridization signal.    -   Thirdly, eventually after a denaturation step, the nucleic acids        present in the sample or the resulting amplified product are        contacted with LiPA strips onto which one or more probes (DNA-,        RNA-, degenerate or modified probes), allowing the detection of        the organisms of interest, are immobilized, and hybridization is        allowed to proceed.    -   Finally, eventually after having performed a wash step, the        hybrids are detected using a convenient and compatible detection        system. From the hybridization signals or patterns observed the        presence or absence of one or several organisms screened for in        that particular biological sample can be deduced.

The amplification system used may be more or less universal, dependingon the specific application needed.

By using universal primers located in the conserved flanking regions ofthe rRNA spacer, i.e. in the 16S gene and the 23S gene, the spacerregion of most if not all organisms of eubacterial origin will beamplified.

For some applications it may be appropriate to amplify not all organismspresent in the sample but more specifically Enterococcus species.

A method of the invention for detection and/or identification ofEnterococcus species in a sample, comprises the steps of:

(i) if need be releasing, isolating and/or concentrating the polynucleicacids present in the sample;

(ii) if need be amplifying the 16S-23S rRNA spacer region, or a part ofit, with at least one suitable primer pair;

(iii) hybridizing the polynucleic acids with at least one probe thathybridizes to the target sequence consisting of SEQ ID NO 1 or 2, or ofthe RNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U, or ofthe complementary form of said SEQ ID NO, or of any homologues, or of afragment of at least 20 contiguous nucleotides thereof;

(iv) detecting the hybrids formed in step (iii);

(v) identification of the micro-organism(s) present in the sample fromthe differential hybridization signals obtained in step (iv).

The part of the ITS mentioned in the step of amplification, is apolynucleotide comprising the target sequence, or the target sequenceitself, the target sequence consisting of SEQ ID NO 1 or 2, or of theRNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U, or of thecomplementary form of said SEQ ID NO 1 or 2, or of any homologues, or ofa fragment of at least 20 contiguous nucleotides thereof.

Preferentially, the present invention provides for a method as describedabove wherein at least 2 micro-organisms are detected simultaneously.

A set of probes as described in step (iii) comprises at least two,three, four, five, six, seven, eight, nine or more probes of theinvention, or equivalents thereof.

In a preferred method of the invention, set of probes as described instep (iii) comprises at least two probes.

Preferred probes are polynucleotides of SEQ ID NO 1 to 84 and theirhomologues.

The present invention also provides for a method as described above,wherein the probes as specified in step (iii) are combined with at leastone other probe, preferentially also from the 16S-23S rRNA spacerregion, enabling the simultaneous detection of different pathogenicbacteria liable to be present in the same sample.

Preferred probes are designed for attaining optimal performance underthe same hybridization conditions so that they can be used in sets forsimultaneous hybridization; this highly increases the usability of theseprobes and results in a significant gain in time and labor.

A kit containing any of the polynucleotides of the present invention isalso an object of the invention.

A kit of the invention comprise the following components:

-   -   at least one polynucleotide hybridizing to the target sequence        consisting of SEQ ID NO 1 or 2, to the RNA form of said SEQ ID        NO 1 or 2 wherein T is replaced by U, to the complementary form        of said SEQ ID NO 1 or 2, or to any of their homologues;    -   a hybridization buffer, or components necessary for producing        said buffer.

A preferred kit comprises

-   -   at least one set of two HybProbes hybridizing, adjacent to each        other with less than 25 nucleotides, preferably less than 5        nucleotides, to the target sequence consisting of SEQ ID NO 1 or        2, to the RNA form of said SEQ ID NO 1 or 2 wherein T is        replaced by U, to the complementary form of said SEQ ID NO 1 or        2, or to any of their homologues;    -   a hybridization buffer, or components necessary for producing        said buffer.

TABLE 1 SEQ ID Preferred NO Name Sequence function 1GTTCATTGAAAACTGGATATTGAAGTAAAAAGAATCAAAACAAACCGAGAACA CCGCGTTGAAT 2GTTCATTGAAAACTGGATATTTGAAGTAAATGTAAGTAATACAAACCGAGAAC ACCGCGTTGAAT 3REncspeFP1V1 5′-ACT-TTG-TTC-AGT-TTT-GAG-AGG-T-3′ PF 4 REncspeFP1V25′-TTT-ACT-TTG-TTC-AGT-TTT-GAG-AGG-3′ PF 5 REncspeFP1V35′-TAC-TTT-GTT-CAG-TTT-TGA-GAG-GT-3′ PF 6 REncspeFP1V45′-TTT-ACT-TTG-TTC-AGT-TTT-GAG-AGG-T-3′ PF 7 REncspeFP1V55′-TTT-ACT-TTG-TTC-AGT-TTT-GAG-AG-3′ PF 8 REncspeFP2V15′-TAC-AAA-CCG-AGA-ACA-CCG-3′ PF 9 REncspeFP3V15′-CCT-CCT-TTC-TAA-GGA-ATA-TCG-G-3′ PF 10 REncspeFP3V25′-CTC-CTT-TCT-AAG-GAA-TAT-CGG-3′ PF 11 5′-GTT-CAT-TGA-AAA-CTG-GAT-A-3′PF 12 REncspeRP1V1 5′-CGG-TGT-TCT-CGG-TTT-GTA-G-3′ PR 13 REncspeRP2V15′-CCT-TCT-TCT-AGC-GAT-AGA-AGG-3′ PR 14 REncspeRP3V15′-ATC-AAC-CTT-ACG-GTT-GGG-3′ PR 15 REncspeRP3V25′-TTA-TCA-ACC-TTG-CGG-TTG-3′ PR 16 REncspeRP3V35′-TTA-TCA-ACC-TTA-CGG-TTG-GGT-3′ PR 17 REncspeRP3V45′-TAT-CAA-CCT-TAC-GGT-TGG-GT-3′ PR 18 REncspeRP4Vl5′-GCA-ATT-GAA-CTT-ATT-AAA-AAA-CTC-3′ PR 19 REncspeRP4V25′-AGC-AAT-TGA-ACT-TAT-TAA-AAA-ACT-C-3′ PR 20 REncspeRP4V35′-GC-AAT-TGA-ACT-TAT-TAA-AAA-AC-3′ PR 215′-ATT-CAA-CGC-GGT-GTT-CTC-G-3′ PR 22 REncfis_cHP2V1.2_5LC65′-TTG-ATT-CTT-TTT-ACT-TCA-ATA-TCC-AGT-TTT-CA-3′ Sensor 23REncfis_cHP2V1.3_5LC6 5′-TTT-TGA-TTC-TTT-TTA-CTT-CAA-TAT-CCA-GTT-TTC-3′Sensor 24 REncfis_cHP2V1_5LC65′-TTT-TGA-TTC-TTT-TTA-CTT-CAA-TAT-CCA-GTT-TTC-AAT- Sensor GAA-CGA-AT-3′25 REncfis_cHP2V2_5LC6 5′-TTT-ACT-TCA-ATA-TCC-AGT-TTT-CAA-TGA-ACG-3′Sensor 26 REncfis_cHP2V3_5LC6 5′-ATC-CAG-TTT-TCA-ATG-AAC-GAA-T-3′ Sensor27 REncfis_HP1_3FL 5′-GGA-ATA-TTA-CGG-AAA-TAC-ACA-TTT-CGT-CTT-T-3′Sensor 28 REncfis_HP2_3FL5′-GAA-AAC-TGG-ATA-TTG-AAG-TAA-AAA-GAA-TCA-AAA-CA-3′ Sensor 29REncfis_HP2V2_3FL 5′-CTG-GAT-ATT-GAA-GTA-AAA-AGA-ATC-AA-3′ Sensor 30REncfis_HP2V3_3FL 5′-TTC-GTT-CAT-TGA-AAA-CTG-GAT-ATT-GAA-GTA-AA-3′Sensor 31 REncfis_HP2V4_3FL 5′-GTT-CAT-TGA-AAA-CTG-GAT-ATT-GAA-GTA-AA-3′Sensor 32 REncfis_HP2V6_3FL5′-CAT-TGA-AAA-CTG-GAT-ATT-TGA-AGT-AAA-AAG-AA-3′ Sensor 33REncfis_HP2V7_3FL 5′-TGA-AAA-CTG-GAT-ATT-TGA-AGT-AAA-AAG-AA-3′ Sensor 34REncfis_HP2V8_3FL 5′-AAC-TGG-ATA-TTG-AAG-TAA-AAA-GAA-TC-3′ Sensor 35REncfis_HP2V9_3FL 5′-GGA-TAT-TGA-AGT-AAA-AAG-AAT-CAA-AAC-3′ Sensor 36REncfis_HP2V10_3FL 5′-CT-GGA-TAT-TGA-AGT-AAA-AAG-AAT-CAA-AAC-3′ Sensor37 REncfum_cHP2V1_5LC6 5′-TAT-TAC-TTA-CAT-TTA-CTT-CAA-ATA-TCC-A-3′Sensor 38 REncfum_cHP2V2_5LC6 5′-TTT-ACT-TCA-AAT-ATC-CAG-TTT-TCA-AT-3′Sensor 39 REncfum_cHP2V3_5LC65′-TAT-CCA-GTT-TTC-AAT-GAA-CAA-ATT-TAA-CAA-CTA-ATG-3′ Sensor 40REncfum_cHP2V3_5LC6 5′-TTT-ACT-TCA-AAT-ATC-CAG-TTT-TCA-ATG-AAC-3′ Sensor41 REncfum_cHP2V4_5LC6 5′-TTT-ACT-TCA-AAT-ATC-CAG-TTT-TCA-ATG-AAC-AA-3′Sensor 42 REncfum_cHP2V5_5LC65′-TAC-TTC-AAA-TAT-CCA-GTT-TTC-AAT-GAA-CAA-3′ Sensor 43REncfum_cHP2V6_5LC6 5′-CAT-TTA-CTT-CAA-ATA-TCC-AGT-TTT-CAA-TGA-ACA-AA-3′Sensor 44 REncfum_HP1_3FL 5′-AAT-ATT-ACG-GAG-ACT-ACA-CAA-TTT-GTT-TTT-3′Sensor 45 REncfum_HP2_3FL 5′-GGA-TAT-TTT-GAA-GTA-AAT-GTA-AGT-AAC-TAC-3′Sensor 46 REncfum_HP2V2_3FL 5′-TGG-ATA-TTT-GAA-GTA-AAT-GTA-AGT-AA-3′Sensor 47 REncfum_HP2V3_3FL 5′-GAT-ATT-TGA-AGT-AAA-TGT-AAG-TAA-3′ Sensor48 REncfum_HP2V4_3FL 5′-TTT-GTT-CAT-TGA-AAA-CTG-GAT-ATT-TGA-AGT-AAA-3′Sensor 49 REncfum_HP2V5_3FL5′-GTT-CAT-TGA-AAA-CTG-GAT-ATT-TGA-AGT-AAA-3′ Sensor 50REncfum_HP2V6_3FL 5′-CAT-TGA-AAA-CTG-GAT-ATT-TGA-AGT-AAA-3′ Sensor 51REncfum_HP2V7_3FL 5′-TTT-GTT-CAT-TGA-AAA-CTG-GAT-ATT-3′ Sensor 52REncfum_HP2V8_3FL 5′-TGA-AAA-CTG-GAT-ATT-TGA-AGT-AAA-3′ Sensor 53REncfum_HP2V9_3FL 5′-GGA-TAT-TTG-AAG-TAA-ATG-TAA-GT-3′ Sensor 54REncfum_HP2V10_3FL 5′-ATT-GAA-AAA-CTG-GAT-ATT-TGA-AGT-AAA-3′ Sensor 55REncfum_HP2V11_3FL 5′-ATT-TGA-AGT-AAA-TGT-AAG-TAA-TAC-3′ Sensor 56REncfum_HP2V12_3FL 5′-GAT-ATT-TGA-AGT-AAA-TGT-AAG-TAA-T-3′ Sensor 57REncfum_HP2V13_3FL 5′-TGA-AAA-CTG-GAT-ATT-TGA-AGT-AAA-TGT-AAG-TA-3′Sensor 58 REncfum_HP2V14_3FL5′-GA-AAA-CTG-GAT-ATT-TGA-AGT-AAA-TGT-AAG-TA-3′ Sensor 59REncspe_cHP2V1_3FL 5′-TTC-AAC-GCG-GTG-TTC-TCG-GTT-T-3′ Anchor 60REncspe_cHP2V2_3FL 5′-CAA-CGC-GGT-GTT-CTC-GGT-TTG-TTT-TGA-TTC-T-3′Anchor 61 REncspe_cHP2V3_3FL5′-TTC-AAC-GCG-GTG-TTC-TCG-GTT-TGT-ATT-ACT-TAC-ATT- Anchor TAC-TTC-AA-3′62 REncspe_cHP2V3_3FL 5′-ATT-CAA-CGC-GGT-GTT-CTC-GGT-TTG-TTT-TGA-TT-3′Anchor 63 REncspe_cHP2V4_3FL 5′-ACG-CGG-TGT-TCT-CGG-TTT-GTT-TTG-ATT-3′Anchor 64 REncspe_cHP2V5_3FL5′-CAA-CGC-GGT-GTT-CTC-GGT-TTG-TTT-TGA-TT-3′ Anchor 65REncspe_cHP2V6_3FL 5′-TTC-AAC-GCG-GTG-TTC-TCG-GTT-TGT-ATT-ACT-T-3′Anchor 66 REncspe_HP1_5LC6 5′-TTT-GTT-CAG-TTT-TGA-GAG-GTT-TAC-TCT-CAA-3′Anchor 67 REncspe_HP2_5LC6 5′-ACC-GAG-AAC-ACC-GCG-TTG-A-3′ Anchor 68REncspe_HP2V2_5LC 5′-ACA-AAC-CGA-GAA-CAC-CGC-GTT-GAA-T-3′ Anchor 69REncspe_HP2V3_5LC 5′-ACA-AAC-CGA-GAA-CAC-CGC-GTT-GAA-T-3′ Anchor 70REncspe_HP2V4_5LC 5′-AGA-ATC-AAA-ACA-AAC-CGA-GAA-CAC-CGC-GTT-G-3′ Anchor71 REncspe_HP2V5_5LC 5′-AAT-CAA-AAC-AAA-CCG-AGA-ACA-CCG-CGT-TGA-AT-3′Anchor 72 REncspe_HP2V6_5LC 5′-ACA-AAC-CGA-GAA-CAC-CGC-GTT-3′ Anchor 73REncspe_HP2V7_5LC 5′-ACC-GAG-AAC-ACC-GCG-TTG-AAT-3′ Anchor 74REncspe_HP2V8_5LC 5′-CC-GAG-AAC-ACC-GCG-TTG-AAT-3′ Anchor 75REncfis_HP2V11_3FL 5′-AC-TGG-ATA-TTG-AAG-TAA-AAA-GAA-TCA-AAA-3′ Sensor76 REncfis_HP2V12_3FL 5′-AAC-TGG-ATA-TTG-AAG-TAA-AAA-GAA-TCA-AAA-3′Sensor 77 REncfis_HP2V13_3FL5′-A-AAC-TGG-ATA-TTc-AAG-TAA-AAA-GAA-TCA-AAA-3′ Sensor 78REncfis_HP2V14_3FL 5′-CT-GGc-TAT-TGA-AGT-AAA-AAG-AAT-CAA-AAC-3′ Sensor79 REncfis_HP2V15_3FL 5′-CT-GGA-TcT-TGA-AGT-AAA-AAG-AAT-CAA-AAC-3′Sensor 80 REncfis_HP2V16_3FL5′-A-AAC-TGG-cTA-TTG-AAG-TAA-AAA-GAA-TCA-AAA-3′ Sensor 81REncfum_HP2V15_3FL 5′-T-TGA-AGT-AAA-TGT-AAG-TAA-TAC-3′ Sensor 82REncfum_HP2V16_3FL 5′-GAT-cTT-TGA-AGT-AAA-TGT-AAG-TAA-T-3′ Sensor 83REncfum_HP2V17_3FL 5′-GAT-ATT-TGc-AGT-AAA-TGT-AAG-TAA-T-3′ Sensor 84REncspe_HP2V9_5LC6 5′-CC-GAG-AAC-ACC-GCG-TTG-AAT-GA-3′ Anchor PF: PrimerForward PR: Primer reverse The function indicated is in fact thepreferred function.

TABLE 2

FP: Forward primers RP: Reverse primer The primer pairs highlighted arepreferred

TABLE 3 Performance in the particular SEQ IDs NO Primercombinationconditions of the examples 28/—/67 28 + —/45/67 28 + 29/—/68 28 +++—/46/68 28 ++ —/47/68 28 ++ 29/46/68 28 + 28/47/68 28 + —/48/69 15 +—/48/69 28 + —/49/69 28 + —/50/69 15 + —/50/69 28 ++ 30/—/69 28 ++30/—/69 15 ++ 31/—/69 28 ++ 30/—/70 15 ++ 31/—/70 15 ++ 30/—/71 15 ++31/—/71 15 ++ 32/—/69 21 ++ 33/—/69 21 + 34/—/69 21 +++ —/48/70 15 +—/49/70 15 + —/50/70 15 + —/48/71 15 + —/49/71 15 + —/50/71 15 + —/53/6921 +++ 32/53/69 21 + —/53/72 21 +++ 34/53/72 21 + 35/—/73 21 +++ —/55/7321 +++ —/56/73 21 +++ 35/55/73 21 ++ 36/56/73 15 +++ 29/—/73 15 +35/—/73 15 + 36/—/73 15 + 75/—/73 15 + 76/—/73 15 + —/46/73 15 + —/47/7315 ++ —/53/73 15 ++ —/55/73 15 ++ —/56/73 15 ++ 35/56/73 15 ++ 77/—/7315 ++ 78/—/73 15 ++ 79/—/73 15 ++ —/81/73 15 ++ —/82/73 15 +++ —/83/7315 + 36/—/84 15 ++ 36/82/73 15 +++

TABLE 4 Enterococcus Clinical Examined Species relevance in this studyE. asini − − E. avium + + E. azikeevi − − E. casseliflavus ++ + E.cecorum + + E. columbae + + E. dispar − − E. durans + + E. faecalis++++ + E. faecium +++ + E. flavescens + − E. gallinarum ++ + E.haemoperoxidus − − E. hirae + + E. malodoratus + + E. moraviensis − − E.mundtii + + E. phoeniculicola − − E. pseudoavium + − E. porcinus − − E.raffinosus + + E. ratti − − E. rottae − − E. saccharolyticus − − E.solitarius − − E. sulfureus − − E. villorum − − Unnamed − (?) −

EXAMPLES

The method used in the examples is a method for the detection ofEnterococci, in particular E. faecalis and/or E. faecium, using theHybProbe system consisting of two Fluorescein-labeled probes of SEQ IDsNO 36 and 56, acting as sensor, and one LC-Red-labeled probe of SEQ IDNO 73 as anchor, in combination with a Enterococcus-genus primer pair ofSEQ IDs NO 5 and 18.

In total a collection of 162 bacterial isolates was used consisting of:

56 Enterococcus faecalis isolates,

50 Enterococcus faecium isolates,

56 strains of different microorganisms.

If the isolates gave not the expected results, the gDNA was retyped byt-RNA PCR (Vaneechoutte, M. et al., 1998, Int. J. Syst. Bacteriol. (48)127-139) and/or the culture was retyped by ApiSTREP (Biomerieux).

The instrumentation is the LightCycler™ (version 1.2) provided with theadequate software (LC-software version 3.5) enabling a Real-Timefluorescence PCR detection.

Example 1 Preparation of the Samples to be Tested

1/. DNA from Pure Cultures

For extracting the DNA from pure cultures, different purificationmethods can be used:

-   -   Lysis with lysostaphin (5 μg/μl) for 1 h at 37° C. and        purification with the QIAamp blood DNA isolation kit (Qiagen)    -   The method of Pitcher et al. (1989)    -   The MagNAPure LC DNA isolation Kit III (Bacteria, Fungi) on the        MagNAPure instrument. Bacterial cells grown O/N on LB plates or        slants were suspended in 100 to 1000 μl TE pH8 for storage at        −20° C. 2 μl to 20 μl was used for extraction according to the        manufacturer's recommendations.    -   QIAamp DNA mini kit (catalog no. 51306-QIAGEN). The culture was        pre-treated enzymatically using lysozyme and lysostaphin.

2/. DNA from Positive Blood Culture Bottles

Blood samples were inoculated in aerobe blood culture bottles(BacT/ALERT FA) and incubated in a BacT/Alert 3D system (OrganonTeknika) at 37° C. until positive. Positivity was monitored by a colorchange from dark green to yellow.

Aliquots (1.5 ml) of the blood cultures were frozen at −70° C. untiluse.

Genomic DNA was prepared as described in the pack insert of the MPLC DNAIsolation Kit III. As recommended for Organon Teknika blood culturebottles, prior to PCR the eluate was centrifuged 10 sec at 14000 rpm tospin down the extracted carbon particles.

Example 2 LightCycler (LC) Protocol

Following the instructions of the manufacturer of the kit LC-FastStartDNA Master Hybridization Probes (cat. No 3 003 248 or No 2 239 272):

-   -   any sample material suitable for PCR in terms of purity,        concentration, and absence of inhibitors can be used;    -   the primers should be at a final concentration of 0.3 to 1 μM        each;    -   the HybProbes at a final concentration of 0.2 μM each, or        double;    -   the concentration of MgCl₂ should be optimized, and may vary        from 1 to 5 mM;    -   and a negative control should be run.

The amplification and melting conditions are described herein after. TheLC software version 3.5 was used. The quantification settings wereF2/back F1 (samples). For the baseline adjustment the arithmetic modewas used. The crossing point (Ct) calculation was based on the secondderivative maximum. The calculation method for the melting peak waspolynomial. The peak area was used to calculate the Tm.

Amplification and melting curve program:

Temp. Slope Acquisition (° C.) Hold time (° C./sec.) mode Denaturation95 10 min 20 None Cycles 95 10 sec 20 None 45x {open oversize brace} 5015 sec 20 SINGLE 72 10 sec 20 None Melting 95 60 sec 20 None 40 60 sec20 None 80 0 sec 0.1 CONTIN- UOUS Cooling 30 0 sec 20 None

Example 3 Results on Purified DNA, Inclusively and Cross ReactivityTests

1/Inclusivity

In total 47 isolates received as E. faecalis were examined. All producedquantification curves (Ct range 15.5-28.4) and 44 had melting peaksaround 58° C. (Tm range 57.4° C.-58.3° C., mean Tm=57.9° C.).

Three out of the 47 isolates received as E. faecalis reacted as E.faecium in the assay, producing melting peaks around 53° C. The resultswith these 3 isolates were confirmed using biochemical identificationtechniques (Api 20Strep, Biomerieux) and tRNA PCR analysis.

Of the 40 isolates received as E. faecium, 37 produced quantificationcurves (Ct range 17.9-29.5) of which 33 produced melting peaks around53° C. (Tm range 51.9° C.-53.2° C., mean Tm=52.7° C.).

The 7 remaining isolates reacted as follows:

-   -   One isolate gave a double peak, one for E. faecalis and one        for E. faecium.    -   Three isolates reacted as E. faecalis with a melting peak around        58° C.    -   Three other isolates gave neither amplification curves nor        melting curves, but reacted positive on gel, so they most        probably belong to the Enterococcus genus, but are non-E.        faecalis/non-E. faecium isolates.

The discrepant samples were analyzed using biochemical identificationtechniques (Api 20Strep, Biomerieux) and tRNA PCR analysis. For all 7samples it was shown that the initial identification was not correct andfor 6 samples the results coincided with the in results of the assay.

2/. Cross-Reactivity

More than 50 different bacterial species were tested (Mycobacteria,Pseudomonas, Streptococci, etc) and also few fungi. None of the testedorganisms generated quantification curves or melting peaks with theassay.

3/. Conclusion:

The results of the inclusivity and crosscheck tests are summarizedbelow. It can be concluded that all E. faecium and E. faecalis isolatesare properly identified and differentiated and that cross-reactivitydoes not occur.

All E. faecalis and/or E. faecium isolates investigated are detected(100% sensitivity) and could be unequivocally distinguished from allother isolates studied.

Summary of the sensitivity and specificity tests.

# of strains with Double # of peak at strains Tm at Tm at Tm <= 53° C.No Taxon tested 53° C. 58° C. 45° C. and 58° C. peak E. faecalis 47 3⁽¹⁾ 44  0 0 0 E. faecium  40* 33   3⁽²⁾ 0  1⁽⁴⁾  3⁽³⁾ Cross-check 56 00 0 0 56  list Human DNA  1 0 0 0 0 1 ⁽¹⁾confirmed E. faecium⁽²⁾confirmed E. faecalis ⁽³⁾confirmed non-E. faecalis/E. faecium⁽⁴⁾re-typing: E. faecalis

Example 4 Results on Blood Cultures

All the 41 positive blood cultures resulted in a positive PCR. Overall,for positive blood culture bottles, the minimum Ct value obtained was 18and the maximum Ct value was 25. The Tm values were very similar, closeto 58° C. for all E. faecalis isolates and close to 53° C. for E.faecium isolates.

In all except one blood cultures the correct pathogen—either E. faecalisor E. faecium—was identified. One positive bottle which was supposed tocontain E. faecium did not produce a growth curve but a melt peak at 45°C. was observed, indicating the presence of an E. durans isolate.Fragment length analysis on gel after universal PCR amplifying thespacer region, resulted in a pattern typical for E. durans and clearlydifferent from E. faecium.

As summarized in the table below, the final assay composition performedin such a way that:

-   -   All E. faecalis and E. faecium are detected (Ct & melting        curves) and differentiated from other organisms including other        Enterococci.    -   There are no cross-reactivities observed with other organisms        from the many different microorganisms tested, or with human        DNA.

F2/Back F1 Peak below Peak at Peak at Ct 50° C. 53° C. 58° C. RESULT +− + − Enterococcus faecium + − + − + − − + Enterococcus faecalis + − − +− + − − Some Enterococci other than − + − − E. faecalis and E. faecium −− − − Negative − − − − Invalid

1. An isolated nucleic acid molecule consisting of SEQ ID NO 1, itscomplementary form and the RNA form thereof.
 2. An isolated nucleic acidmolecule consisting of SEQ ID NO 2, its complementary form and the RNAform thereof.
 3. An isolated nucleic acid molecule of more than 10contiguous nucleotides that specifically hybridizes to SEQ ID NO 1 or 2,or to the RNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U,or to the complementary form of said SEQ ID NO 1 or 2, or to a fragmentof at least 20 contiguous nucleotides thereof, or to any of theirhomologues, for the detection and/or identification of Enterococcusspecies.
 4. An isolated nucleic acid molecule according to claim 3consisting of a nucleic acid selected from the group consisting of SEQID NO 22 to 26, 28 to 43, 45 to 65 and 67 to
 84. 5. A set of two orthree polynucleotide probes which hybridize to the same target sequencein adjacent locations on said target sequence, said probes hybridizingspecifically to SEQ ID NO 1 or SEQ ID NO 2 or homologues, or to theirRNA form wherein T is replaced by U, or to their complementary form,wherein there are no more than 25 nucleotides between said probes alongsaid target sequence.
 6. A set of two or three polynucleotide probesaccording to claim 5 consisting of any combinations of Table
 3. 7. Acomposition comprising at least one nucleic acid molecule according toclaim 1 and/or a set of two polynucleotide probes, said probescomprising more than 10 contiguous nucleotides.
 8. A method of detectingand/or identification of Enterococcus species in a sample comprisinghybridizing a nucleic acid molecule of claim 3 to nucleic acid sequencesof said sample and detecting said hybridization.
 9. The method of claim8 wherein said Enterococcus species is at least one of E. faecalis andE. faecium.
 10. A method according to claim 8 for detection and/oridentification of Enterococcus species in a sample comprising the stepsof: (i) if need be releasing, isolating and/or concentrating thepolynucleic acids in the sample; (ii) if need be amplifying the 16S-23SrRNA spacer region, or a fragment comprising a Enterococcusspecies-specific polynucleic acid, with at least one suitable primerpair; (iii) hybridizing the polynucleic acids of step (i) or (ii) withat least one polynucleotide probe of claim 3, (iv) detecting the hybridsformed, and (v) interpreting the signal(s) obtained and inferring thepresence of Enterococcus species and/or identifying the Enterococcusspecies in the sample.
 11. A method according to claim 10 wherein asuitable primer pair consists any combination of a forward primerpolynucleotide selected from the group consisting of SEQ ID NO 3, 4, 5,6, 7, 8, 9, 10 or 11 and their homologues, and a reverse primerpolynucleotide selected from the group consisting of SEQ ID NO 12, 13,14, 15, 16, 17, 18, 19, 20 or 21 and their homologues.
 12. A methodaccording to claim 10 wherein said at least one probe comprises twopolynucleotide probes.
 13. A method according to claim 12 wherein saidat least one probe comprises a set of two polynucleotide probes whichhybridize to the same target sequence in adjacent locations on saidtarget sequence, wherein there are no more than 25 nucleotides betweensaid probes along the hybridized polynucleic acid sequence.
 14. A methodaccording to claim 12 wherein the two polynucleotide probes consist ofany combination of polynucleotides of Table
 3. 15. A kit for detectionand/or identification of Enterococcus species comprising the followingcomponents: at least one nucleic acid molecule according to claim 3, anda hybridization buffer, or components necessary for producing saidbuffer.